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Abstract:

A combination of a BCR-ABL inhibitor and a hedgehog pathway inhibitor for
the treatment of leukemia.

Claims:

1. A combination comprising a first agent that is a Smoothened inhibitor
and a second agent that is a BCR-ABL inhibitor, wherein the first agent
is a compound of Formula I: ##STR00011## in which Y1 and Y2
are independently selected from N and CR10; wherein R10 is
selected from hydrogen, halo, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and --OXNR10aR10b; wherein
R10a and R10b are independently selected from hydrogen and
C1-6alkyl; R1 is selected from cyano, halo, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy, C6-10aryl, dimethyl-amino,
C1-6alkyl-sulfanyl and C3-8heterocycloalkyl optionally
substituted with up to 2 C1-6alkyl radicals; R2 and R5 are
independently selected from hydrogen, cyano, halo, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and dimethylamino; R3 and R4
are independently selected from hydrogen, halo, cyano, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy and
halosubstituted-C1-6alkoxy; or either R1 and R2 or R1
and R5 together with the phenyl to which they are both attached form
C5-10heteroaryl; R6 and R7 are independently selected from
hydrogen, C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy
and halosubstituted-C1-6alkoxy; with the proviso that R6 and
R7 are not both hydrogen; R8 is selected from hydrogen, halo,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy and
halosubstituted-C1-6alkoxy; R9 is selected from
--S(O)2R11, --C(O)R11, --OR11, --NR12aR12b
and --R11; wherein R11 is selected from aryl, heteroaryl,
cycloalkyl and heterocycloalkyl; R12a and R12b are
independently selected from C1-6alkyl and
hydroxy-substituted-C1-6alkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl of R9 can be optionally substituted
with 1 to 3 radicals independently selected from C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy, C6-10aryl-C0-4alkyl,
C5-10heteroaryl-C0-4alkyl, C3-12cycloalkyl and
C3-8heterocycloalkyl; wherein said aryl-alkyl substituent of R9
is optionally substituted with 1 to 3 radicals independently selected
from halo, C1-6alkyl, halosubstituted-C1-6alkyl,
C1-6alkoxy, halosubstituted-C1-6alkoxy and methyl-piperazinyl;
or a pharmaceutically acceptable salt thereof or a compounds of the
formula (II): ##STR00012## and pharmaceutically acceptable salts
thereof, wherein R1 is a C6-14 aryl group, or a 5-14 membered
heteroaryl group which may be unsubstituted or substituted; R2 and R3 are
independently C1-8 alkyl, C1-8 alkylOH, or R2 and R3 form a
fused C3-14 cycloalkyl group; L is a bond, C1-8 alkylene,
--C(O)O--, --C(O)NR9--, --C1-8 alkylOH--, --C1-8haloalkyl-,
--C(O)--, --NH-- or --O--; X and W are independently N or CR5, and at
least one of X or W is N; R7 is a C6-14 aryl group, a 5-14 membered
heteroaryl group, or a 3-14 membered cycloheteroalkyl group; R4 is
C1-8 alkyl, C2-8 alkenyl, C3-14 cycloalkyl, a C6-14
aryl group, a 5-14 membered heteroaryl group, a 3-14 membered
cycloheteroalkyl group, C1-8 alkoxy, halo, NR6R8, C(O)OR6,
C(O)NR6R8, C1-8haloalkyl, formyl, carbalkoxy, C1-8alkylOH,
C(O)R6, SO2R6, C(O)NHC1-8alkylR6, NR6R8, SO2NR6R8,
OCF3, NHC(O)R6, CH2OC(O)NR6R8, CH2NR6R8, NHC(O)OR6,
NHC(O)NR6R8, CH2NHSO2R6, CH2NHC(O)OR6, OC(O)R6, or
NHC(O)R6, which may be substituted or unsubstituted; Z is C1-8
alkyl, CN, OH, or halogen; m and p are independently 0-3; Y is a bond,
C1-8 alkylene, --C(O)--, --C(O)O--, --CH(OH)--, or --C(O)NR10; R5 is
H, halogen, CN, lower alkyl, OH, OCH3 or OCF3; Wherein R1 may
be substituted by one or more of C1-8 alkyl, a C6-14 aryl
group, C1-8 haloalkyl, C1-8 alkoxy, halo, NH2, CN,
OCF3, OH, C(O)NR6R8, C(O)R6, NR6R8, NHC(O)R6, SO2R6,
SO2NR6R8; R9 and R10 are independently C1-8 alkyl or H; R6 and
R8 are independently H, C1-8 alkyl, C2-8 alkenyl, C3-14
cycloalkyl, a C6-14 aryl group, a 5-14 membered heteroaryl group, a
3-14 membered cycloheteroalkyl group, C1-8haloalkyl, C1-8
alkylOH, C1-8alkoxy, or two R6 on one atom can form a heteroatom
containing ring; and wherein R4, R6, and R8 can be unsubstituted or
substituted by one or more of C1-8 alkyl, C3-14 cycloalkyl, a
C6-14 aryl group, a 5-14 membered heteroaryl group, a 3-14 membered
cycloheteroalkyl group, C1-8 alkylOH, OH, oxo, C1-8 haloalkyl,
carboxC1-8 alkyl, or SO2C1-8alkyl, halo, --OCH3,
--OCF3, --OH, --NH2 or a salt thereof.

2. The combination of claim 1, wherein said first agent is
2-methyl-4'-trifluoromethoxy-biphenyl-3-carboxylic acid
[6-(cis-2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide or a
pharmaceutically acceptable salt thereof.

3. The combination of claim 1, wherein said first agent is
2-[(R)-4-(6-benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahyd-
ro-2H-[1,2]bipyrazinyl-5'-yl]-propan-2-ol or a pharmaceutically acceptable
salt thereof.

4. The combination of claim 1, wherein said second agent is an ABL
inhibitor, an ABL/Scr inhibitor, an Aurora kinase inhibitor, or a non-ATP
competitive inhibitor of BCR-ABL.

7. The combination of claim 6, wherein the first agent is
2-methyl-4'-trifluoromethoxy-biphenyl-3-carboxylic acid
[6-(cis-2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide or a
pharmaceutically acceptable salt thereof.

8. The combination of claim 6, wherein the first agent is
2-[(R)-4-(6-benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahyd-
ro-2H-[1,2']bipyrazinyl-5'-yl]-propan-2-ol or a pharmaceutically
acceptable salt thereof.

9. A pharmaceutical composition comprising a first agent that is a
Smoothened inhibitor and a second agent that is a BCR-ABL inhibitors,
wherein the first agent is a compound of Formula I: ##STR00013## in
which Y1 and Y2 are independently selected from N and
CR10; wherein R10 is selected from hydrogen, halo,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and --OXNR10aR10b; wherein
R10a and R10b are independently selected from hydrogen and
C1-6 alkyl; R1 is selected from cyano, halo, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy, C6-10aryl, dimethyl-amino,
C1-6alkyl-sulfanyl and C3-8heterocycloalkyl optionally
substituted with up to 2 C1-6alkyl radicals; R2 and R5 are
independently selected from hydrogen, cyano, halo, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and dimethylamino; R3 and R4
are independently selected from hydrogen, halo, cyano, C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy and
halosubstituted-C1-6alkoxy; or either R1 and R2 or R1
and R5 together with the phenyl to which they are both attached form
C5-10heteroaryl; R6 and R7 are independently selected from
hydrogen, C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy
and halosubstituted-C1-6alkoxy; with the proviso that R6 and
R7 are not both hydrogen; R8 is selected from hydrogen, halo,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy and
halosubstituted-C1-6alkoxy; R9 is selected from
--S(O)2R11, --C(O)R11, --OR11, --NR12aR12b
and --R11; wherein R11 is selected from aryl, heteroaryl,
cycloalkyl and heterocycloalkyl; R12a and R12b are
independently selected from C1-6alkyl and
hydroxy-substituted-C1-6alkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl of R9 can be optionally substituted
with 1 to 3 radicals independently selected from C1-6alkyl,
halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy, C6-10aryl-C0-4alkyl,
C5-10heteroaryl-C0-4alkyl, C3-12cycloalkyl and C3-8
heterocycloalkyl; wherein said aryl-alkyl substituent of R9 is
optionally substituted with 1 to 3 radicals independently selected from
halo, C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and methyl-piperazinyl; or a
pharmaceutically acceptable salt thereof or a compounds of the formula
(II): ##STR00014## and pharmaceutically acceptable salts thereof,
wherein R1 is a C6-14 aryl group, or a 5-14 membered heteroaryl
group which may be unsubstituted or substituted; R2 and R3 are
independently C1-8 alkyl, C1-8 alkylOH, or R2 and R3 form a
fused C3-14 cycloalkyl group; L is a bond, C1-8 alkylene,
--C(O)O--, --C(O)NR9--, --C1-8 alkylOH--, --C1-8 haloalkyl-,
--C(O)--, --NH-- or --O--; X and W are independently N or CR5, and at
least one of X or W is N; R7 is a C6-14 aryl group, a 5-14 membered
heteroaryl group, or a 3-14 membered cycloheteroalkyl group; R4 is
C1-8 alkyl, C2-8 alkenyl, C3-14 cycloalkyl, a C6-14
aryl group, a 5-14 membered heteroaryl group, a 3-14 membered
cycloheteroalkyl group, C1-8 alkoxy, halo, NR6R8, C(O)OR6,
C(O)NR6R8, C1-8haloalkyl, formyl, carbalkoxy, C1-8alkylOH,
C(O)R6, SO2R6, C(O)NHC1-8alkylR6, NR6R8, SO2NR6R8,
OCF3, NHC(O)R6, CH2OC(O)NR6R8, CH2NR6R8, NHC(O)OR6,
NHC(O)NR6R8, CH2NHSO2R6, CH2NHC(O)OR6, OC(O)R6, or
NHC(O)R6, which may be substituted or unsubstituted; Z is C1-8
alkyl, CN, OH, or halogen; m and p are independently 0-3; Y is a bond,
C1-8 alkylene, --C(O)--, --C(O)O--, --CH(OH)--, or --C(O)NR10; R5 is
H, halogen, CN, lower alkyl, OH, OCH3 or OCF3; Wherein R1 may
be substituted by one or more of C1-8 alkyl, a C6-14 aryl
group, C1-8 haloalkyl, C1-8 alkoxy, halo, NH2, CN,
OCF3, OH, C(O)NR6R8, C(O)R6, NR6R8, NHC(O)R6, SO2R6,
SO2NR6R8; R9 and R10 are independently C1-8 alkyl or H; R6 and
R8 are independently H, C1-8 alkyl, C2-8 alkenyl, C3-14
cycloalkyl, a C6-14 aryl group, a 5-14 membered heteroaryl group, a
3-14 membered cycloheteroalkyl group, C1-8haloalkyl, C1-8
alkylOH, C1-8alkoxy, or two R6 on one atom can form a heteroatom
containing ring; and wherein R4, R6, and R8 can be unsubstituted or
substituted by one or more of C1-8 alkyl, C3-14 cycloalkyl, a
C6-14 aryl group, a 5-14 membered heteroaryl group, a 3-14 membered
cycloheteroalkyl group, C1-8 alkylOH, OH, oxo, C1-8 haloalkyl,
carboxC1-8 alkyl, or SO2C1-8alkyl, halo, --OCH3,
--OCF3, --OH, --NH2 or a salt thereof.

11. The composition of claim 10, wherein the second agent is nilotinib.

12. The composition of claim 11, wherein the first agent is
2-methyl-4'-trifluoromethoxy-biphenyl-3-carboxylic acid
[6-(cis-2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide or a
pharmaceutically acceptable salt thereof.

13. The composition of claim 11, wherein the first agent is
2-[(R)-4-(6-benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahyd-
ro-2H-[1,2']bipyrazinyl-5'-yl]-propan-2-ol or a pharmaceutically
acceptable salt thereof.

Description:

[0001] This is a continuation of application Ser. No. 12/539,855 filed on
Aug. 12, 2009, which in its entirety is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] A combination of a BCR-ABL inhibitor and a hedgehog pathway
inhibitor for the treatment of leukemia.

[0004] 2. Related Background Art

[0005] The Hedgehog signaling pathway has been described in the art (see,
e.g., Nybakken et al., Curr. Opin. Genet. Dev. 2002, 12:503-511; and Lum
et al., Science 2003, 299: 2039-2045). Briefly, in the absence of
hedgehog ligands, the transmembrane receptor, Patched (Ptch), binds to
Smoothened (Smo) and blocks Smo's function. This inhibition is relieved
in the presence of ligands, which allows Smo to initiate a signaling
cascade that results in the release of transcription factors Glis from
cytoplasmic proteins fused (Fu) and Suppressor of Fused (SuFu). In the
inactive situation, SuFu prevents Glis from translocating to the nucleus.
In the active situation, Fu inhibits SuFu and Glis are released. Gli
proteins translocate into the nucleus and control target gene
transcription.

[0007] BCR-ABL has been shown to induce proliferation and anti-apoptosis
through various mechanisms in committed myeloid or lymphoid progenitors
or 3T3 fibroblasts. (Pendergast et al., Cell 1993, 75:175-85; Ilaria et
al., J. Biol. Chem. 1996, 271:31704-10; Chai et al., J. Immunol. 1997,
159:4720-8; and Skorski et al., EMBO J. 1997, 16:6151-61). However,
little is known about the effect of BCR-ABL on the hematopoietic stem
cell (HSC) population. Recent publications suggest that developmental
pathways like the Wnt signaling pathway or the Polycomb gene BMI1 might
be involved in the regulation and expansion of leukemic stem cells (Mohty
et al., Blood, 2007; Hosen et al., Stem Cells, 2007). BMI1 and
beta-catenin are both upregulated in CML blast crisis and their
expression correlates with the progression of the disease. BCR-ABL
positive granulocyte-macrophage progenitors that have acquired
β-catenin expression are candidate leukemic stem cells in
blast-crisis CML. Self-renewal pathways are involved in the expansion of
the BCR-ABL positive leukemic stem cell during chronic phase, which leads
to the initial expansion of the malignant clone.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention provides combinations and therapeutic methods of
treatment which may be useful for inhibiting tumor cell growth and for
treating a variety of cancers.

[0009] In one aspect, the present invention provides a combination
comprising a first agent that inhibits the hedgehog signaling pathway and
a second agent that inhibits BCR-ABL. In another aspect, the invention
provides pharmaceutical compositions comprising a therapeutically
effective amount of a first agent that inhibits hedgehog signaling
pathway, a second agent that inhibits BCR-ABL, and a pharmaceutically
acceptable carrier.

[0010] The invention also provides methods for treating cancers,
particularly a BCR-ABL positive leukemia, such as CML, comprising
administering to a system or a subject, a therapeutically effective
amount of a composition comprising a first agent that inhibits hedgehog
signaling pathway and a second agent that inhibits BCR-ABL, or
pharmaceutically acceptable salts or pharmaceutical compositions thereof,
thereby treating said BCR-ABL positive leukemia. For example, the
compositions of the invention may be used to treat chronic myeloid
leukemia or acute lymphocyte leukemia.

[0011] Furthermore, the present invention provides for the use of a
therapeutically effective amount of a combination comprising a first
agent that inhibits hedgehog signaling pathway and a second agent that
inhibits BCR-ABL, or pharmaceutically acceptable salts or pharmaceutical
compositions thereof, in the manufacture of a medicament for treating a
cell proliferative disorder, particularly BCR-ABL positive leukemia.

[0012] In the above compositions and methods for using the compositions of
the invention, the first agent in the inventive composition may bind to
Smo. In other embodiments, the second agent in the inventive composition
is an ABL inhibitor, an ABL/Scr inhibitor, an Aurora kinase inhibitor, or
a non-ATP competitive inhibitor of BCR-ABL.

[0013] In the above combinations, compositions and methods for using the
compositions of the invention, the inventive composition may be
administered to a system comprising cells or tissues. In some
embodiments, the invention composition may be administered to a human
patient or animal subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows a "first replate" experiment in which the total number
of secondary colonies derived from a single colony of primary CML cells
formed after 3 days treatment with increasing concentrations of Compound
A. Results are expressed as a percentage of untreated control.

[0015] FIG. 2 shows total colonies of primary CML cells formed following a
"second replate" of FIG. 1. Results are expressed as a percentage of
untreated control. shows total colonies formed following a second
replate.

[0016] FIG. 3 describes the total numbers of resultant secondary colonies
as a percentage of the untreated control in three replicates.

[0017] FIG. 4 is an illustrative example of the total numbers of secondary
colonies in a experiment of primary CML cells treated with compound A or
nilotinib or the two drugs in combination for 3 days. Results are
expressed as a percentage of the untreated control.

[0018] FIG. 5 indicates the total number of secondary colonies produced in
"first replate" experiments following 7 days exposure to compound A,
nilotinib or a combination of the two. Results are expressed as a
percentage of the untreated control.

[0019] FIG. 6 indicates the total number of secondary colonies produced in
"first replate" experiments following 3 days exposure to compound A,
nilotinib or a combination of the two. Results are expressed as a
percentage of the untreated control.

[0020] FIG. 7 demonstrates the proliferation index (PI) of primary CML
cells after treatment with compound A, nilotinib or both drugs by
calculating the area under the curve (AUC) for the assays. The PI
reflects both the colonies produced and their extinction rate.

[0021] FIG. 8 is 2.5×105 mouse bone marrow cells infected with
Bcr-abl retrovirus were plated in 400 ul per well (48-well plate) in
OPTI-MEM media (10% FBS, 0.1% 2-Mercaptoethanol, 50 ng/ml SCF, 25 ng/ml
mIL-3 and 25 ng/ml mIL-6) in the presence of the indicated concentrations
of AMN107 and compound A. After 3 days of culture cells were plated in
methylcellulose at a concentration of 1500 cells per 35 mm plate. Colony
formation was scored 10 days after plating.

[0022] FIG. 9. Colonies obtained in the 1st plating experiment of
FIG. 8 were resuspended and washed in PBS containing 10% FCS. Cells were
resuspended in OPTI-MEM media and plated in methylcellulose at a
concentration of 5000 cells per 35 mm plate. Colony formation was scored
10 days after plating.

[0023] FIG. 10 compares survival rates in a mouse CML model with a control
vehicle, Compound A, AMN107, and a combination of Compound A and ANM107.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is further exemplified, but not limited, by
the following representative examples, which are intended to illustrate
the invention and are not to be construed as being limitations thereon.

[0025] Compounds for Formula I--Smoothened Inhibitors

[0026] In one aspect, the present invention provides a compound of Formula
I:

##STR00001##

[0027] in which

[0028] Y1 and Y2 are independently selected from N and
CR10; wherein R10 is selected from hydrogen, halo,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and --OXNR10aR10b; wherein
R10a and R10b are independently selected from hydrogen and
C1-6alkyl;

[0031] R3 and R4 are independently selected from hydrogen, halo,
cyano, C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy
and halosubstituted-C1-6alkoxy; or either R1 and R2 or
R1 and R5 together with the phenyl to which they are both
attached form C5-10heteroaryl;

[0032] R6 and R7 are independently selected from hydrogen,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy and
halosubstituted-C1-6alkoxy; with the proviso that R6 and
R7 are not both hydrogen;

[0034] R9 is selected from --S(O)2R11, --C(O)R11,
--OR11, --NR12aR12b and --R11; wherein R11 is
selected from aryl, heteroaryl, cycloalkyl and heterocycloalkyl;
R12a and R12b are independently selected from C1-6alkyl
and hydroxy-substituted-C1-6alkyl;

[0036] wherein said aryl-alkyl substituent of R9 is optionally
substituted with 1 to 3 radicals independently selected from halo,
C1-6alkyl, halosubstituted-C1-6alkyl, C1-6alkoxy,
halosubstituted-C1-6alkoxy and methyl-piperazinyl; and the N-oxide
derivatives, prodrug derivatives, protected derivatives, individual
isomers and mixture of isomers thereof; and the pharmaceutically
acceptable salts and solvates (e.g. hydrates) of such compounds.

[0037] In a second aspect, the present invention provides a pharmaceutical
composition which contains a compound of Formula I or a N-oxide
derivative, individual isomers and mixture of isomers thereof; or a
pharmaceutically acceptable salt thereof, in admixture with one or more
suitable excipients.

[0060] In another embodiment, the present invention includes compounds of
formula (II) wherein R4 is

##STR00007##

which may be unsubstituted or substituted.

[0061] In another embodiment, the present invention includes compounds of
formula (II) wherein R2 and R3 are C1-8 alkyl.

[0062] In a still further embodiment, the present invention includes
compounds of formula (II) wherein R2 and R3 are CH3.

[0063] In another embodiment, the present invention includes compounds of
formula (II) wherein L is --O--, --NH--, --C(O)--, --CH(OH)--,
--CH2--, --CF2--, --CHF--, --COH--, or a bond. In another
embodiment, the present invention includes compounds of formula (I)
wherein L is --CH2--. In another embodiment, the present invention
includes compounds of formula (I) wherein both X are N, and Z is
CH3.

[0064] In another embodiment, the present invention includes a compound of
formula (IIa):

[0069] Compounds of Formula II and IIa are further described in the
contents of U.S. patent application Ser. No. 12/503,565, which has
counterpart International Application No. PCT/EP09/059,138.

[0070] A preferred compound of formula (II) is
2-[(R)-4-(6-Benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahyd-
ro-2H-[1,2']bipyrazinyl-5'-yl]-propan-2-ol, (also identified as Compound B
in this document), of the below formula:

##STR00009##

2-[(R)-4-(6-Benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahyd-
ro-2H-[1,2']bipyrazinyl-5'-yl]-propan-2-ol can be made according to Scheme
1

##STR00010##

First Step:

[0071] A mixture of 4,5-dimethyl-1,4-dichloro-pyridazine (10 g, 56.5
mmol), tetrakis(triphenylphosphine)palladium(0) (3.3 g, 2.80 mmol) and
THF (200 mL) is degassed and then benzylzinc bromide (147 mL, 0.5 M in
THF, 73.40 mmol) is added. The reaction solution is heated to 65°
C. overnight. Solvent is removed. Water is added and the water layer is
extracted with EtOAc. The organic layer is concentrated to afford a crude
product that is purified by chromatography on silica gel (EtOAc/Heptane:
0%˜50%) to give 3-benzyl-6-chloro-4,5-dimethyl-pyridazine_(9.5 g,
67%).

[0074] To a solution of
(R)-4-(6-benzyl-4,5-dimethyl-pyridazin-3-yl)-2-methyl-3,4,5,6-tetrahydro--
2H-[1,2']bipyrazinyl-5'-carboxylic acid methyl ester (840 mg, 1.85 mmol)
in THF (12 mL) is added methyl magnesium bromide (5 mL, 15 mmol, 3M in
ether) at -78° C. The reaction mixture is stirred at 0° C.
for 2 h then diluted with DCM and washed with NH4Cl and water. The
combined organic layers are washed with water, brine, dried over
Na2SO4, filtered and concentrated down. Purification by HPLC of
the crude product with acetonitrile in water (from 10% to 95% with 3%
1-propanol) at 220 nm wavelength detection provides the desired compound
B (400 mg, 50%) next to small amounts of the corresponding methyl ketone.
The solvents are removed with a lyophilizer to provide the products as
white powders.

[0077] "Alkyl" as a group and as a structural element of other groups, for
example halo-substituted-alkyl and alkoxy, can be either straight-chained
or branched. C1-4-alkoxy includes, methoxy, ethoxy, and the like.
Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl, and
the like.

[0079] "Heteroaryl" is as defined for aryl above where one or more of the
ring members is a heteroatom. For example C5-10heteroaryl is a
minimum of 5 members as indicated by the carbon atoms but that these
carbon atoms can be replaced by a heteroatom. Consequently,
C5-10heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl,
quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl,
benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl,
oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

[0081] "Heterocycloalkyl" means cycloalkyl, as defined in this
application, provided that one or more of the ring carbons indicated, are
replaced by a moiety selected from --O--, --N═, --NR--, --C(O)--,
--S--, --S(O)-- or --S(O)2--, wherein R is hydrogen, C1-4alkyl
or a nitrogen protecting group. For example, C3-8heterocycloalkyl as
used in this application to describe compounds of the invention includes
morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl,
piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, thiomorpholino,
sulfanomorpholino, sulfonomorpholino, etc.

[0082] "Halogen" (or halo) preferably represents chloro or fluoro, but may
also be bromo or iodo.

[0083] The term "agent" or "test agent" includes any substance, molecule,
element, compound, entity, or a combination thereof. It includes, but is
not limited to, e.g., protein, polypeptide, small organic molecule,
polysaccharide, polynucleotide, and the like. It can be a natural
product, a synthetic compound, a chemical compound, or a combination of
two or more substances. Unless otherwise specified, the terms "agent",
"substance", and "compound" can be used interchangeably.

[0084] As used herein, "contacting" has its normal meaning and refers to
combining two or more molecules (e.g., a small molecule organic compound
and a polypeptide) or combining molecules and cells (e.g., a compound and
a cell). Contacting can occur in vitro, e.g., combining two or more
agents or combining a compound and a cell or a cell lysate in a test tube
or other container. Contacting can also occur in a cell or in situ, e.g.,
contacting two polypeptides in a cell by coexpression in the cell of
recombinant polynucleotides encoding the two polypeptides, or in a cell
lysate.

[0085] The term "hedgehog" is used to refer generically to any member of
the hedgehog family, including sonic, indian, desert and tiggy winkle.
The term may be used to indicate protein or gene. The term is also used
to describe homolog/ortholog sequences in different animal species.

[0086] The terms "hedgehog (Hh) signaling pathway" and "hedgehog (Hh)
signaling" are used interchangeably and refer to the chain of events
normally mediated by various members of the signaling cascade such as
hedgehog, patched (Ptch), smoothened (Smo), and Gli. The hedgehog pathway
can be activated even in the absence of a hedgehog protein by activating
a downstream component. For example, overexpression of Smo will activate
the pathway in the absence of hedgehog.

[0087] Hh signaling components or members of Hh signaling pathway refer to
gene products that participate in the Hh signaling pathway. An Hh
signaling component frequently affects the transmission of the Hh signal
in cells/tissues, typically resulting in changes in degree of downstream
gene expression level and/or phenotypic changes. Hh signaling components,
depending on their biological function and effects on the final outcome
of the downstream gene activation/expression, may be divided into
positive and negative regulators. A positive regulator is an Hh signaling
component that positively affects the transmission of the Hh signal,
i.e., stimulates downstream biological events when Hh is present.
Examples include hedgehog, Smo, and Gli. A negative regulator is an Hh
signaling component that negatively affects the transmission of the Hh
signal, i.e., inhibits downstream biological events when Hh is present.
Examples include (but are not limited to) Ptch and SuFu. Smo is an
essential component of the Hh signaling pathway.

[0088] Hedgehog signaling antagonists, antagonists of Hh signaling or
inhibitors of Hh signaling pathway refer to agents that inhibit the
bioactivity of a positive Hh signaling component (such as hedgehog, Ptch,
or Gli) or down-regulate the expression of the Hh signaling component.
They also include agents which up-regulate a negative regulator of Hh
signaling component. A hedgehog signaling antagonist may be directed to a
protein encoded by any of the genes in the hedgehog pathway, including
(but not limited to) sonic, indian or desert hedgehog, smoothened,
ptch-1, ptch-2, gli-1, gli-2, gli-3, etc.

[0089] The terms "inhibit," "inhibiting" or "inhibition," in the context
of modulation of enzymatic activities, inhibition relates to reversible
suppression or reduction of an enzymatic activity including competitive,
uncompetitive, and noncompetitive inhibition. This can be experimentally
distinguished by the effects of the inhibitor on the reaction kinetics of
the enzyme, which may be analyzed in terms of the basic Michaelis-Menten
rate equation. Competitive inhibition occurs when the inhibitor can
combine with the free enzyme in such a way that it competes with the
normal substrate for binding at the active site. A competitive inhibitor
reacts reversibly with the enzyme to form an enzyme-inhibitor complex
[EI], analogous to the enzyme-substrate complex.

[0090] The term "subject" includes mammals, especially humans. It also
encompasses other non-human animals such as cows, horses, sheep, pigs,
cats, dogs, mice, rats, rabbits, guinea pigs, monkeys. The term "patient"
refers to a human patient.

[0091] The term "treating" includes the administration of compounds or
agents to prevent or delay the onset of the symptoms, complications, or
biochemical indicia of a disease (e.g., leukemia), alleviating the
symptoms or arresting or inhibiting further development of the disease,
condition, or disorder. Treatment may be prophylactic (to prevent or
delay the onset of the disease, or to prevent the manifestation of
clinical or subclinical symptoms thereof) or therapeutic suppression or
alleviation of symptoms after the manifestation of the disease.

[0092] Pharmacology and Utility

[0093] The combination of the present invention may be used for treating a
variety of cancers. In one embodiment, the invention provides an agent
that inhibits the hedgehog signaling pathway in combination with an agent
that inhibits BCR-ABL, for inhibiting the growth and proliferation of
hematopoietic tumors of lymphoid lineage including leukemia, acute
lymphocytic leukemia (ALL), acute lymphoblastic leukemia, B-cell
lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma,
hairy cell lymphoma, histiocytic lymphoma, and Burkitts lymphoma; and
hematopoietic tumors of myeloid lineage including acute and chronic
myelogenous leukemias (CML), myelodysplastic syndrome, myeloid leukemia,
and promyelocytic leukemia.

[0094] The combination of the present invention are also useful for
treating cancers known to be associated with protein tyrosine kinases
such as, for example, Src, BCR-ABL and c-kit. In particular embodiments,
the combination of the present invention are useful for treating cancers
that are sensitive to and resistant to chemotherapeutic agents that
target BCR-ABL and c-kit. In particular embodiments, the combination of
the present invention may be used for treating BCR-ABL-positive CML and
ALL.

[0095] Chronic myelogenous leukemia (CML) is a cancer of the bone marrow
characterized by increased and unregulated clonal proliferation of
predominantly myeloid cells in the bone marrow. Its annual incidence is
1-2 per 100,000 people, affecting slightly more men than women. CML
represents about 15-20% of all cases of adult leukemia in Western
populations, about 4,500 new cases per year in the U.S. or in Europe.
(Faderl et al., N. Engl. J. Med. 1999, 341: 164-72).

[0096] CML is a clonal disease that originates from a single transformed
hematopoietic stem cell (HSC) or multipotent progenitor cell (MPP)
harboring the Philadelphia translocation t(9/22). The expression of the
gene product of this translocation, the fusion oncogene BCR-ABL, induces
molecular changes which result in expansion of the malignant
hematopoiesis including the leukemic stem cell (LSC) pool and the
outgrowth and suppression of non-malignant hematopoiesis (Stam et al.,
Mol Cell Biol. 1987, 7:1955-60). Myeloid cells (granulocytes, monocytes,
megakaryocytes, erythrocytes), but also B- and T-cells express BCR-ABL,
indicating the MPP or HSC as the start point of the disease. (Fialkow et
al., J. Clin. Invest. 1978, 62:815-23; Takahashi et al., Blood 1998,
92:4758-63). In contrast to oncogenes causing AML, like MOZ-TIF2 or
MLL-ENL, BCR-ABL does not confer self-renewal properties to committed
progenitor cells, but rather utilizes and enhances the self-renewal
properties of existing self-renewing cells, like HSCs or MPPs. During the
course of the disease, the leukemic stem cell pool expands and in the
final stage, the blast crisis, nearly all CD34+CD38- cells carry the
Philadelphia translocation.

[0097] Imatinib mesylate (STI571, GLEEVEC®) is the standard of therapy
for CML with response rates of more than 96%, and works by inhibiting the
activity of BCR-ABL. However, despite initial success, patients
eventually develop resistance to imatinib mesylate due to acquisition of
point mutations in BCR-ABL. In view of the limitations of imatinib
mesylate, there is a need for improved methods for treating CML.

[0098] In addition, it is contemplated that the combination of the present
invention may be used for treating carcinoma including that of the
bladder (including accelerated and metastatic bladder cancer), breast,
colon (including colorectal cancer), kidney, liver, lung (including small
and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate,
testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas
(including exocrine and endocrine pancreatic carcinoma), esophagus,
stomach, gall bladder, cervix, thyroid, and skin (including squamous cell
carcinoma); tumors of the central and peripheral nervous system including
astrocytoma, neuroblastoma, glioma, medulloblastoma and schwannomas;
tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma,
and osteosarcoma; and other tumors including melanoma, Merkel cell
carcinoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid
follicular cancer, and teratocarcinoma. It is also contemplated that the
combinations of the present invention may be used for treating
mastocytosis, germ cell tumors, pediatric sarcomas, and other cancers.

[0099] The therapeutic methods described herein may be used in combination
with other cancer therapies. For example, Hh antagonists in combination
with BCR-ABL inhibitors may be administered adjunctively with any of the
treatment modalities, such as chemotherapy, radiation, and/or surgery.
For example, they can be used in combination with one or more
chemotherapeutic or immunotherapeutic agents; and may be used after other
regimen(s) of treatment is concluded. Examples of chemotherapeutic agents
which may be used in the compositions and methods of the invention
include but are not limited to anthracyclines, alkylating agents (e.g.,
mitomycin C), alkyl sulfonates, aziridines, ethylenimines,
methylmelamines, nitrogen mustards, nitrosoureas, antibiotics,
antimetabolites, folic acid analogs (e.g., dihydrofolate reductase
inhibitors such as methotrexate), purine analogs, pyrimidine analogs,
enzymes, podophyllotoxins, platinum-containing agents, interferons, and
interleukins.

[0101] The present methods may be used to treat primary, relapsed,
transformed, or refractory forms of cancer, including the development of
resistance, such as mutations in BCR-ABL leading to resistance. Often,
patients with relapsed cancers have undergone one or more treatments
including chemotherapy, radiation therapy, bone marrow transplants,
hormone therapy, surgery, and the like. Of the patients who respond to
such treatments, they may exhibit stable disease, a partial response
(i.e., the tumor or a cancer marker level diminishes by at least 50%), or
a complete response (i.e., the tumor as well as markers become
undetectable). In either of these scenarios, the cancer may subsequently
reappear, signifying a relapse of the cancer.

[0102] In accordance with the foregoing, the present invention further
provides a method for preventing or treating any of the diseases or
disorders described above in a subject in need of such treatment, which
method comprises administering to said subject a therapeutically
effective amount (See, "Administration and Pharmaceutical Compositions",
infra) of a compound of Formula I or a pharmaceutically acceptable salt
thereof. For any of the above uses, the required dosage will vary
depending on the mode of administration, the particular condition to be
treated and the effect desired.

[0103] Administration and Pharmaceutical Compositions:

[0104] In general, compounds of the invention will be administered in
therapeutically effective amounts via any of the usual and acceptable
modes known in the art, either singly or in combination with one or more
therapeutic agents. A combination of the present invention includes
administration at same time as well as sequential administration. A
therapeutically effective amount may vary widely depending on the
severity of the disease, the age and relative health of the subject, the
potency of the compound used and other factors. An indicated daily dosage
in the larger mammal, e.g. humans, is in the range from about 10 mg to
about 2,500 mg, more preferably about 100 mg to 1000 mg, in dosages such
as 100 mg, 200 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg and
1000 mg. These dosages can be conveniently administered, e.g. in divided
doses up to four times a day or in retard form. Suitable unit dosage
forms for oral administration comprise from ca. 1 to 50 mg active
ingredient.

[0105] Compounds of the invention can be administered as pharmaceutical
compositions by any conventional route, in particular enterally, e.g.,
orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,
in the form of injectable solutions or suspensions, topically, e.g., in
the form of lotions, gels, ointments or creams, or in a nasal or
suppository form. Pharmaceutical compositions comprising a compound of
the present invention in free form or in a pharmaceutically acceptable
salt form in association with at least one pharmaceutically acceptable
carrier or diluent can be manufactured in a conventional manner by
mixing, granulating or coating methods. For example, oral compositions
can be tablets or gelatin capsules comprising the active ingredient
together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,
stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;
for tablets also c) binders, e.g., magnesium aluminum silicate, starch
paste, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose and or polyvinylpyrrolidone; if desired d)
disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or
effervescent mixtures; and/or e) absorbents, colorants, flavors and
sweeteners. Injectable compositions can be aqueous isotonic solutions or
suspensions, and suppositories can be prepared from fatty emulsions or
suspensions. The compositions may be sterilized and/or contain adjuvants,
such as preserving, stabilizing, wetting or emulsifying agents, solution
promoters, salts for regulating the osmotic pressure and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. Suitable formulations for transdermal applications include an
effective amount of a compound of the present invention with a carrier. A
carrier can include absorbable pharmacologically acceptable solvents to
assist passage through the skin of the host. For example, transdermal
devices are in the form of a bandage comprising a backing member, a
reservoir containing the compound optionally with carriers, optionally a
rate controlling barrier to deliver the compound to the skin of the host
at a controlled and predetermined rate over a prolonged period of time,
and means to secure the device to the skin. Matrix transdermal
formulations may also be used. Suitable formulations for topical
application, e.g., to the skin and eyes, are preferably aqueous
solutions, ointments, creams or gels well-known in the art. Such may
contain solubilizers, stabilizers, tonicity enhancing agents, buffers and
preservatives.

[0106] Compounds of the invention can be administered in therapeutically
effective amounts in combination with one or more therapeutic agents
(pharmaceutical combinations). For example, synergistic effects can occur
with immunomodulatory or anti-inflammatory substances or other anti-tumor
therapeutic agents. Where the compounds of the invention are administered
in conjunction with other therapies, dosages of the co-administered
compounds will of course vary depending on the type of co-drug employed,
on the specific drug employed, on the condition being treated and so
forth.

[0107] The invention also provides for a pharmaceutical combinations, e.g.
a kit, comprising a) a first agent which is a compound of the invention
as disclosed herein, in free form or in pharmaceutically acceptable salt
form, and b) at least one co-agent. The kit can comprise instructions for
its administration.

[0108] The terms "co-administration" or "combined administration" or the
like as utilized herein are meant to encompass administration of the
selected therapeutic agents to a single patient, and are intended to
include treatment regimens in which the agents are not necessarily
administered by the same route of administration or at the same time.

[0109] The term "pharmaceutical combination" as used herein means a
product that results from the mixing or combining of more than one active
ingredient and includes both fixed and non-fixed combinations of the
active ingredients. The term "fixed combination" means that the active
ingredients, e.g. a compound of Formula I and a co-agent, are both
administered to a patient simultaneously in the form of a single entity
or dosage. The term "non-fixed combination" means that the active
ingredients, e.g. a compound of Formula I and a co-agent, are both
administered to a patient as separate entities either simultaneously,
concurrently or sequentially with no specific time limits, wherein such
administration provides therapeutically effective levels of the 2
compounds in the body of the patient. The latter also applies to cocktail
therapy, e.g. the administration of 3 or more active ingredients.

[0110] A compound of the invention can be prepared as a pharmaceutically
acceptable acid addition salt by reacting the free base form of the
compound with a pharmaceutically acceptable inorganic or organic acid.
Alternatively, a pharmaceutically acceptable base addition salt of a
compound of the invention can be prepared by reacting the free acid form
of the compound with a pharmaceutically acceptable inorganic or organic
base.

[0111] Alternatively, the salt forms of the compounds of the invention can
be prepared using salts of the starting materials or intermediates.

[0112] The free acid or free base forms of the compounds of the invention
can be prepared from the corresponding base addition salt or acid
addition salt from, respectively. For example a compound of the invention
in an acid addition salt form can be converted to the corresponding free
base by treating with a suitable base (e.g., ammonium hydroxide solution,
sodium hydroxide, and the like). A compound of the invention in a base
addition salt form can be converted to the corresponding free acid by
treating with a suitable acid (e.g., hydrochloric acid, etc.).

[0113] Compounds of the invention in unoxidized form can be prepared from
N-oxides of compounds of the invention by treating with a reducing agent
(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,
sodium borohydride, phosphorus trichloride, tribromide, or the like) in a
suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueous
dioxane, or the like) at 0 to 80° C.

[0114] Prodrug derivatives of the compounds of the invention can be
prepared by methods known to those of ordinary skill in the art (e.g.,
for further details see Saulnier et al., (1994), Bioorganic and Medicinal
Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs
can be prepared by reacting a non-derivatized compound of the invention
with a suitable carbamylating agent (e.g.,
1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the
like).

[0115] Protected derivatives of the compounds of the invention can be made
by means known to those of ordinary skill in the art. A detailed
description of techniques applicable to the creation of protecting groups
and their removal can be found in T. W. Greene, "Protecting Groups in
Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999.

[0116] Compounds of the present invention can be conveniently prepared, or
formed during the process of the invention, as solvates (e.g., hydrates).
Hydrates of compounds of the present invention can be conveniently
prepared by recrystallization from an aqueous/organic solvent mixture,
using organic solvents such as dioxin, tetrahydrofuran or methanol.

[0117] Compounds of the invention can be prepared as their individual
stereoisomers by reacting a racemic mixture of the compound with an
optically active resolving agent to form a pair of diastereoisomeric
compounds, separating the diastereomers and recovering the optically pure
enantiomers. While resolution of enantiomers can be carried out using
covalent diastereomeric derivatives of the compounds of the invention,
dissociable complexes are preferred (e.g., crystalline diastereomeric
salts). Diastereomers have distinct physical properties (e.g., melting
points, boiling points, solubilities, reactivity, etc.) and can be
readily separated by taking advantage of these dissimilarities. The
diastereomers can be separated by chromatography, or preferably, by
separation/resolution techniques based upon differences in solubility.
The optically pure enantiomer is then recovered, along with the resolving
agent, by any practical means that would not result in racemization. A
more detailed description of the techniques applicable to the resolution
of stereoisomers of compounds from their racemic mixture can be found in
Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and
Resolutions", John Wiley And Sons, Inc., 1981.

[0118] One of skill in the art will appreciate that the above
transformations are only representative of methods for preparation of the
compounds of the present invention, and that other well known methods can
similarly be used.

[0120] Viable cells were enumerated using trypan blue dye exclusion and
set up in culture in serum free medium (SFM) with the stated
concentrations of Compound A and/or nilotinib. Following 72 hours (h)
culture the cells were washed twice in phosphate buffered solution (PBS)
and viable cells counted, again by trypan blue exclusion. These cells
were then used for a series of colony forming and re-plating assays.

[0121] In the colony forming assay (CFA) a single cell suspension is
created in semisolid media with appropriate cytokines. This enables the
assessment of colony formation from each individual cell. In order to
measure the effect of compound A and/or nilotinib on the relative
abundance of CD34+ CP CML progenitor cells, CFA were set up in METHOCULT
with cells plated at an initial concentration of 4000 cells per mL in
duplicate. Colonies were identified and enumerated 14-16 days (d)
following plating.

[0122] An accepted in vitro technique to approximate self-renewal activity
is serial re-plating. Colonies derived from a CFA are individually
plucked and re-dispersed in further METHOCULT. The capacity to reform
colonies following re-dispersion is related to the number of primitive
progenitors remaining within each individual colony and is therefore an
indirect measurement of self-renewal. Following CFA 20-30 individual
non-erythroid colonies from each experimental arm were then plucked with
a p10 pipettor (one tip per colony using an inverted microscope) and
carefully dispersed into 100 μL METHOCULT with a further 10 μL SFM
in 96 well plates prior to incubation for a further 7d. Resultant
secondary colonies were enumerated in each well and; in the case of wells
containing secondary colonies; the entire contents were re-dispersed in a
further 100 μL METHOCULT to assess tertiary colony formation.

[0123] Colony assays were performed in METHOCULT with cells plated at an
initial concentration of 4000 cells per mL in duplicate. Colonies were
identified and enumerated 14-16 days (d) following plating. 20-30
individual non-erythroid colonies from each experimental arm were then
plucked with a p10 pipettor (one tip per colony using an inverted
microscope) and carefully dispersed into 100 μL METHOCULT with a
further 10 μL SFM in 96 well plates prior to incubation for a further
7d. Resultant secondary colonies were enumerated in each well and; in the
case of wells containing secondary colonies; the entire contents were
re-dispersed in a further 100 μL METHOCULT to assess tertiary colony
formation.

[0124] FIG. 1 indicates the total resultant secondary colonies following
the first replating as a percentage of the untreated control in three
replicates (error bars indicate the standard error of the mean (SEM)).
FIG. 2 illustrates the total number of tertiary colonies formed following
second re-plate. These figures indicate a reduction in re-plating
capacity with compound A alone and in combination with nilotinib and this
is consistent with an inhibition of self-renewal behaviour in the treated
cells.

Example 2

[0125] Colony forming assays (CFAs) were performed as described above.
Colonies were identified and enumerated 14-16d following plating. 20-30
individual, non-erythroid colonies from each experimental arm were then
plucked as above and carefully dispersed into 100 μL METHOCULT with a
further 10 μL SFM in 96 well plates prior to incubation for a further
7d. Resultant secondary colonies were enumerated in each well. FIG. 3
describes the total numbers of resultant secondary colonies as a
percentage of the untreated control in three replicates (significance was
assessed by unpaired 2 tailed t test).) and indicates a reduction in
re-plating capacity consistent with an inhibition of self-renewal
behavior in the treated cells.

Example 3

[0126] Combination experiments were performed on primary CD34+selected
chronic phase (CP) CML cells. Following thaw and overnight culture
primary cells were exposed to Compound A at varying concentrations with
or without co-exposure to nilotinib for 72 h in SFM. CFAs and subsequent
re-plating experiments were conducted as previously detailed. FIG. 4
details the total numbers of secondary colonies as a percentage of the
untreated control in one illustrative example.

Example 4

[0127] Combination experiments were performed on primary CD34+selected CP
CML cells. Following thaw and overnight culture these primary cells were
cultured in Compound A at a final concentration of 10 nM and/or nilotinib
at a final concentration of 5 μM for 7d with CFAs and subsequent
re-plating performed as previously indicated. FIG. 5 indicates the total
number of secondary colonies produced following re-plating in 3
experiments (error bars indicate the SEM and significance was determined
by unpaired 2 tailed t test). FIG. 5 demonstrates a reduction in
re-plating capacity in these cells following exposure to nilotinib and
compound A in combination for 7d.

Example 5

[0128] Combination experiments were performed on primary CD34+selected CP
CML cells. Following thaw and overnight culture these primary cells were
cultured in Compound A at a final concentration of 10 nM and/or nilotinib
at a final concentration of 5 μM for 3d with CFAs and subsequent
re-plating performed as previously indicated. FIG. 6 indicates the total
number of secondary colonies produced following re-plating in 4
experiments (error bars indicate the SEM and significance was determined
by unpaired 2 tailed t test). FIG. 6 indicates a non-significant increase
in re-plating capacity in these cells following 3d treatment with
nilotinib and a reduction in re-plating capacity following 3d exposure to
nilotinib and compound A.

Example 6

[0129] Another measure of the degree of occurring is to assess the
proliferation index (PI) of re-plated colonies. The fate of each
re-plated colony is to either become extinct or to produce a number (n)
secondary colonies. 20-30 individual non-erythroid colonies from each
experimental arm were then plucked with a p10 pipettor (one tip per
colony using an inverted microscope) and carefully dispersed into 100
μL METHOCULT with a further 10 μL SFM in 96 well plates prior to
incubation for a further 7d. Resultant secondary colonies were enumerated
in each well and; in the case of wells containing secondary colonies; the
entire contents were re-dispersed in a further 100 μL METHOCULT to
assess tertiary colony formation. The PI is a measure of self-renewal
that reflects both number of colonies produced and the overall extinction
rate. The inverse cumulative distribution of secondary colonies is
assessed by graphing the proportion of wells with greater than n colonies
and calculating the resultant area under the curve (AUC). In FIG. 7 the
PI has been assessed from the resultant totals of all colonies re-plated
in each experimental arm in 4 separate experiments (3d nilotinib and/or
compound A exposure) with a total of 132 colonies per arm. This figure
demonstrates a relative increase in PI following nilotinib therapy as
compared to untreated cells and a reduction in PI following treatment
with compound A alone and in combination with nilotinib.

Example 7

[0130] Mouse bone marrow cells were infected with a bicistronic retroviral
Bcr-Abl vector (Bcr-Abl-IRES-GFP). Infected bone marrow cells were
cultured for 72 h in the presence of cytokines and different
concentrations of Compound A or AMN107, and then plated in
methylcellulose. No reduction of colony formation was seen in the first
methylcellulose plating for the groups pre-treated with AMN107 or
Compound A compared to the control DMSO group (FIG. 8). However, a
pronounced reduction in colony number was detected in the groups
pre-treated with Compound A upon secondary re-plating of the colonies
(FIG. 9). Clonogenic colony formation assays assess the self-renewal
capacity of early progenitor/stem cells. Data indicate that Compound A
inhibits the clonogenic capacity of Bcr-Abl transformed mouse CML bone
marrow cells.

Example 8

[0131] A bone marrow transplant model of Bcr-Abl was used to induce CML in
mice. Briefly, a bicistronic retroviral Bcr-Abl vector (Bcr-Abl-IRES-GFP)
was used to produce virus to infect progenitor bone marrow (BM) cells
collected from mice previously treated with 5-FU. After 3 rounds of
infection, 200,000 GFP positive progenitor BM cells were transplanted
into lethally irradiated hosts. Two weeks post-BMT, peripheral blood
samples were analyzed by FACS analysis to establish the percentage of GFP
positive cells present in the recipient mice. The 32 mice included in the
study had 10-20% of GFP positive cells. 14 days post-BMT, the mice were
stratified into 4 groups of 8 animals each and received a two week
treatment with Vehicle, compound A at 80 mg/kg po qd, AMN107 75 mg/kg po
qd or the combination between Compound A and AMN107. During the entire
study period the mice were followed for any sign of leukemia development,
such as hunched position, lost of body weight or inability of grooming.
The animals were sacrificed when they reached any of the previously
described signs.

[0132] As shown in FIG. 10, all the vehicle and compound A treated animals
were sacrificed between 18 to 56 days post-BMT, in the AMN107 treated
group 5 animals were sacrificed to date and only 3 mice were sacrificed
in the combination group. This suggests an advantage in the combination
of these compounds for the treatment of CML.